S.F. Marotta, L.G. Hiles, D.M. Lanuza and U. Boonayathap
Hanruz, P.E., O'Dea, R.F. and N.D. Goldberg: Phosphodiesterase inhibition by papaverine and structurally related compound5. Biochem.Pharmacol. 21: 2266-2268 (1972) Ho, R.J. and E. W. Sutherland: Formation and release of a hormone antagonist by rat adipocytes. J.BioI.Chem. 246: 6822-6827 (1971) Ingebretsen, c., J.F. Clark, D.O. Allen and J. Ashmore: Effect of glucagon, dibutyryl cyclic 3',5'-AMP and phosphodiesterase inhibitors on rat liver phosphorylase activity and cyclic 3',5'-AMP levels. Biochem.Pharmacol. 23: 2139-2146 (1974) Ingebretsen, W.R., Jr. and S.R. Wagle: A rapid method for the isolation of large quantities of rat liver parenchymal cells with high anabolie rates. Biochem.Biophys.Res. Comm. 47: 403-410 (1972) Johnson, M.E.M., N.M. Das, F.R. Butcher and J.N. Fain: The regulation of gluconeogenesis in isolated rat liver eells by glucagon, insulin, dibutyryl cyclic adenosine monophosphate and fatty acids. J.BioI.Chem. 247: 3229· 3235 (1972)
Kono, T.: Destruction and restoration of the insulin effector system of isolated fat cells. J.BioI.Chem. 244: 57775784 (1969) Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. RandalI: Protein measurement with the folin phenol reagent. J.BioI.Chem. 193: 265-275 (1951) Manganiello, V.C., F. Murad and M. Vaughan: Affects of lipolytic and antilipolytic agents on cyclic 3',5'-adenosine monophosphate in fat cells. J.BioI.Chem. 246: 21952202 (1971) Pock, G. and W.R. Kukovetz: Papaverine-induced inhibition of phosphodiesterase activity in various marnmalian tissue. Life Sei. I. Physiol.Pharmacol. 10: 133-144 (1971) fohl, S.L., H.M.J. Krans, L. Birnbaumer and M. Rodbell: Inactivation of glucagon by plasma membranes of rat liver. J.BioI.Chem. 247: 2295-2301 (1972) Sutherland, E. W. and T. W. Rall: Fractionation and characterization of a cyclic adenosine ribonucleotide formed by tissue particles. J.BioI.Chem. 232: 1077-1091 (1958)
Requests for reprints should be addressed to: Dr. D.O. Allen, Department of Pharmacology, Indiana University, School of Medicine, 1100 West Michigan Street, Indianapolis, Indiana 46202 (USA)
Horm. Metab. Res. 7 (1975) 334-337
© Georg Thieme Verlag Stuttgart
The Relation of Hepatic In Vitro I nactivation of Corticosteroids to the Circadian Rhythm of Plasma Corticosterone* S.F. Marotta, L.G. Hiles, D.M. Lanuza and U. Boonayathap** Research Resources Center and the Department of General Nursing, University of Illinois at the Medical Center, Chicago, Illinois, U.S.A.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
334
Summary
Introduction
Plasma corticosterone levels and the in vitro capacity of the liver to reduce the 6,4 3-ketone group of corticosterone were ascertained at 4 hr intervals in male rats maintained on a normal lighting schedule (12L: 120). The rates of 6,4 3-ketone reduction, as weil as wet liver weight, were highest during the early portion (08.00 hr) of the light period when liver protein and plasma corticosterone concentrations were low. Shortly (2000 hr) after the beginning of the dark period plasma corticosterone reached peak levels, while hepatic inactivation of corticosterone was markedly depressed. This inverse relationship suggests that the rhythmicity in the capacity of the liver to inactivate corticosterone may contribute to the circadian periodicity of plasma corticosterone.
Although circadian rhythms in plasma adrenocortical steroid concentrations have been weH documented in human beings (Samuels, Browll, Eik-Nes, Tyler and Dominguez 1957, Lanuza and Marotta 1974) and lower animals (Ungar and Halberg 1962, Hiroshige, Sakakura and ftoh 1969, Marotta, Lanuza and Hiles 1974), investigators continue to search at various levels of the hypothalamo-hypophyseal-adrenocortical (HHA) system for the mechanisms which initiate or affect this periodicity. Thus, circadian r:-,ythms have been reported for a~ the uptake of corticosterone (lIß, 21-dihydroxy-4-pregnene-3,20-dione) by the hippocampus which is considered a possible negative feedback site (Stevens, Reed, Erickson and Grosser 1973), b) proposed neurotransmitters (biogenic amines) for the corticotropin-releasing hormone (CRH) in the central nervous system (Friedman and Walker 1968, Scapagnini, Moberg, VanLoon, DeGroot and Ganong 1971), c) hypothalamic content of CRH (Hiroshige, Sakakura and ftoh 1969, Retiene and Schulz 1970), d) concentrations of adrenocorticotropin (ACTH) in the adenohypophysis and plasma
Key·Words: Circadian Plasma Corticosterone - Hepatic Steroid Inactivation
·Supported in part by internal grants of the University of lllinois and by the Office of Naval Research contract NR 201-020 "Present address: Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand Received: 13 Jan. 1975
Accepted: 21 Apr. 1975
335
(Retiene and Schulz 1970, Gallagher, Yoshida, Rolf warg, Fukushima, Weitzman and Hel/man 1973), e) in vivo and in vitro responses of the adrenal cortex to the administration of ACTH (Ungar and Halberg 1962, Andrews 1968) and f) in vitro production of steroids by adrenal quarters in the absence of ACTH (Ungar and Halberg 1962, Andrew 1968).
at 06.00 hr from the cages of animals sacrificed from 08.00 to 16.00 hr, and at 18.00 hr for those sacrificed from 20.00 to 04.00 hr. The animals were decapitated in less than 15 seconds after removal from their cages in the constant temperature room. Trunk blood, which was collected in heparinized tubes, was immediately centrifuged and the plasma stored at -20 oC until analyzed for corticosterone by the fluorometric method of Guillemin, Clayton, Lipscomb and Smith (1959).
The periodicity in plasma steroid levels is dependent upon not only those HHA mechanisms which govern the cyclic release of adrenocortical steroids, bu t also those mechanisms wh ich affect the metabolism and renal excretion of steroids. Circadian variations in the latter have been correlated with the diurnal changes in glomerular filtration rate (Markei, Sharp, Slorach and Vipond 1962, Marotta and Linwong 1966), which in turn may influence the amounts of steroid found in urine (Boonayathap and Marotta 1974). Studies on whole body metabolism of adrenocortical steroids, as assessed by the plasma disappearance rate (t 1/2) following rapid infusion of pharmacologic loads of cortisol (IIß, 17, 21-trihydroxy-4-pregnene-3, 20-dione) have not shown diurnal differences (Perkol!. Eik-Nes, Nugent, Fred. Nimer, Rush, Samuels and Tyler 1959). On the other hand, deLacerda, Kowarski and Migeon (1973), using radio labelled cortisol, have reported diurnal variations in the metabolic clearance rate of this steroid. Since Samuels et al. (1957) suggested that the t 1/2 is affected by the initial cortisol concentration, one could postulate that the circadian alterations in plasma cortisol levels would also modify the t 1/2 of this steroid. However, the disappearance of cortisol from plasma is affected primarily by the rate at which this steroid is inactivated by the liver such that the in vitro enzymatic activity (e.g. t,4 -steroid hydrogenase ) of the Iiver is closely associated with the in vivo changes in t 1/2 (Herbst, Yates, Glenister and Urquhart 1960). Although the rates of hepatic inactivation of adrenocortical steroids have been shown to vary when animals are subjected to various stressors (Herbst et al. 1960, Silah 1971), no attempt has been made to ascertain whether the liver exhibits a circadian periodicity in its enzymatic capacity to inactivate these steroids. Thus a study was designed to a) determine the in vitro rate of reduction of corticosterone by the liver of male rats sacrificed every 4 hr throughout a 24 hr period, and b) correlate this reduction rate with the weil known circadian rhythm in plasma corticosterone levels.
The entire liver was rapidly removed from the animals and placed in ice cold iso tonic saline. The Iiver was biotted dry, weighed and a portion (approximately 500 mg) homogenized in a Sorval Omni-Mixer with a solution containing sucrose (0.25 M) and nicotinamide (0.04 M) such that the final concentration of the homogenate was 20% w/v. The homogenate was centrifuged at 2000 RPM for 15 min at 4 0 C. Protein concentration (Lowry, Rosebrough, Fa" and Randall 1951) and the capacity of the liver to inactivate corticosterone. as measured by the reduction rate of the t,4 3ketone group (Glenn, Stafford, Lyster and Bowman 1957), were determined in the supernatant fraction. All the necessary cofactors (magnesium chloride, phosphate buffer at pH 7.4. sucrose, nicotinamide, glucose-6-phosphate, trisodium iso citrate and nicotinamide adenine dinucleotide phosphate) as prescribed by Glenn et al. (1957) were present in the incubation medium together with 100 pg corticosterone which served as the substrate. After one hr incubat ion at 370 C the homogenate was extracted with purified cold methylene chloride and the Iatter phase was washed successively with 0.1 N NaOH, 0.1 N HCI and water. Aliquots of the methylene chloride extract were evaporated to dryness at 35 0 C under reduced pressure (-10 Ib/sq.in). The residue was dissolved in 5.0 ml methyl alcohol, transferred to matched quartz cuvettes and read at 240 nm in a Beckman DBG spectrophotometer. Appropriate tissue blanks incubated at OOC and standards were also carried throughout the entire procedure.
Materials and Methods Forty-two male rats (Holtzman Co., Madison, Wis.) were placed 7/cage in a constant temperature room (21 oC) for approximately two weeks with a lighting schedule of 12L: 12D with light from 06.00 to 18.00 hr. Seven rats were sacrificed by decapitation every 4 hr beginning at 08.00 hr and ending the following morning at 04.00 hr. Food (Purina rat chow), which had been fed ad libitum, was removed
Results The mean body weights of rats sacrificed at 4 hr intervals throughout the day were not significantly different from each other, while the liver weights (gm/IOO gm B.W.) of rats sacrificed frorn 08.00 to 16.00 hr were significantly heavier (10 to 45%) than those found in animals from 20.00 to 04.00 hr (Table 1). These changes in liver weight were probably due to changes in water content, since soon after the beginning (06.00 hr) of the light period the hepatic protein concentrations were considerably lower (110 to 115 mg/gm L.W.) than the peak values (137 to 144 mg/gm L.W.) noted toward the end of the dark period. In general, the capacity of the liver to reduce the t,4 3-ketone group, whether reported as the rate of corti~osterone disappearance per gram wet liver (f..Lg Cort./gm L.W./hr) or per gram liver protein (mg Cort./gm L.P./hr), was significantly greater during the light (06.00 to 18.00 hr) than during the dark (18.00 to 06.00 hr) period. The capacity of the liver to inactivate corticosterone began to gradually decline from peak disappearance rates (1.63 mg/gm L.P./hr) at 08.00 hr to minimal rates (0.77 mg/gm L.P./hr) at 24.00 hr before beginning to rise in the
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Hepatic In Vitro Inactivation of Corticosteroids
336
S.F. Marotta, L.G. Hiles, D.M. Lanuza and U. Boonayathap
Table 1. Relation of plasma corticosterone levels to hepatic weight, pro tein content and 6 4 3-ketone reduction rate of male rats on a schedule of light from 06.00 to 18.00 hr and dark from 18.00-06.00 hr Time of Day (hr) 12.00
16.00
20.00
24.00
04.00
Body Weight (B.W.) gm
227'" (213-242)
217 (204-231)
233 (224-243)
227 (221-234)
215 (201-228)
228 (219-238)
Liver Weight (L.W.) gm/IOO gm B.W.
4.9 (4.6-5.3)
4.8 (4.6-5.0)
4.5 (4.3-4.6)
3.9 (3.6-4.1 )
3.8 (3.7-3.9)
3.3 (3.2-3.4 )
6 4 3-Ketone Reduction
174 (165-182)
175 (150-200)
165 (154-178)
118 (106-130)
106 (90-123)
118 (75-163)
UO (100-120)
115 (111-119)
131 (128-135)
133 (130-135)
137 (132-142)
144 (140-149)
mg Cort./gm L.P./hr
1.63 (1.48-1.78)
1.55 (1.31-1.79)
1.25 (1.17-1.33)
0.90 (0.81-0.98)
0.83 0.77 (0.62-0.91 ) (0.53-1.13 )
Plasma Corticosterone pg/lOO ml
5.0 (3.3-6.8)
5.6 (4.3-6.9)
14.2 (11.9-16.6)
21.7 (15.2-28.1)
14.3 (10.5-18.0)
Cort./gm L.W./hr Liver Protein (L.P.) mg/gm L.W.
l1g
6 4 3-Ketone Reduction
7.9 (6.2-9.7)
"'Mean with 95% confidence limits in ( ).
next 4 hr (Table l). During this time the plasma corticosterone pattern, which exhibited basal levels (5.0 to 5.9 /Jg/loo ml) in the morning and significantly higher levels (14.2 to 2l.7 /Jg/lOO ml) from 16.00 to 24.00 hr, were inversely (r = 0.51; P < 0.05) related to the rates of hepatic inactivation of corticosterone. Although the time (08.00 to 12.00 hr) for minimal plasma corticosterone levels and maximal hepatic inactivation coincided, peak corticosterone levels (20.00 hr) were observed approximately 4 hr prior to the nadir of hepatic in vitro inactivation of corticosterone.
inactivation of steroids may not only be mediated via the release of adrenocortical steroids, but also to its direct effect on inhibiting the hepatic enzymes involved in reducing ring A of the corticosteroids
(Komel 1970).
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Measurements
In addition to the above factors which affect the hepatic inactivation of adrenocortical steroids, Herbst et al. (1960) showed that variations in food intake markedly affect the activity of this hepatic enzyme system, and hence any stressor which diminishes food intake depresses the capacity of the liver to inactivate adrenocortical steroids. Diurnal studies on food intake have shown that rats on a normal lightDiscussion ing schedule consume more food from 18.00 to Many investigators have reported circadian variations 24.00 hr than at other times during the day (Herbst in the activities of hepatic enzymes involved in the et al. 1960). To partially avoid this factor in the metabolism of cholesterol (VanCantfort 1974), tyro- present study, rats sacrificed from 08.00 to 16.00 hr si ne (Cohn, Joseph, Larin, Shoemaker and Wurtman did not have access to food from 06.00 hr, while those 1970) and tryptophan (Rapoport, Feigin, Bruton sacrificed from 20.00 to 04.00 hr were withou t food and Beisel 1966). Furthermore, these enzymatic acfrom 18.00 hr. When comparing groups of rats durtivities are related to the circulating levels of adreno- ing the light period with those du ring the dark period cortical steroids since adrenalectomy modified their but without access to food for the same length of circadian periodicity. Hypo- and hyperadrenocortitime, the rates of reduction of the 6 4 3-ketone group were always higher during the light period than thc calism also appear to affect the hepatic capacity to dark period. It is unlikely, therefore, that the periodreduce the 6 4 _3 ketone group. Urquhart, Yates and Herbst (1959) have shown that severe adrenocortiicity observed in hepatic inactivation of corticosterone cal insufficiency in rats with an accompanying loss is markedly affected by the circadian rhythm in food in body weight caused a marked depression in hepatic intake, since minimal hepatic activity was observed reduction rates, while unilateral adrenalectomy with- during aperiod of normally high food intake. out a loss in body weight or bilateral adrenalectomy The data presented herein suggest that the sustained in rats maintained on saline (Hagen and Troop 1960), elevation in plasma corticosterone levels, and presum~aused no c~ange or a ~light in.cre~e, respect.ively, ably ACTH as weIl, which preceded minimal in vitro m the capacIty of the hver to machv.ate corhcost~rone. hepatic inactivation of corticosterone, may contribute On ~he other hand, pretreatment ~f l~tact rats ~lt.h to the depression in hepatic enzymatic activity; and cOr~lSO?e or ACT~ depre~sed the zn vltro hepahc m- in turn the latter would then cause a gradual increase in plasma corticosterone levels which would inhibit actlvahon of corhcosterOlds (Urquhart, Yates an~ Herbst 1959, Hagen and Troop 1960). Recent eVlthe release of ACTH. Thus, it is conceivable that the dence suggests that the effects of ACTH on hepatic
Hepatic In Vitro Inactivation of Corticosteroids
daily variations observed in plasma eortieosteroid eoneentrations may not only be due to rhythms previously shown in hypothalamie CRH (JIiroshige, Sakakura and Itoh 1969), adenohypophyseal and plasma ACTH (Retiene and Schulz 1970, Gal/agher
337
et al. 1973), adrenoeortieal seeretion (Andrews 1968), and the metabolie clearanee of eortieosteroids (de Lacerda, Kowarski and Migeon 1973), but also to the diurnal eapaeity of the liver to inaetivate these steroids. Lowry, O.H., N.J. Rosebrough, A.L. Farr, R.F. Randall: Protein measurement with Folin-phenol re agent. J.biol. Chem. 193: 265-275 (1951) Martel, P.J., G. W.G. Sharp, S.A. Slorach, H.J. Vipond: A study of the roles of adrenocortical steroids and glomerular filtration rate in the mechanism of the diurnal rhythm of water and electrolyte excretion. J.Endocr. 24: 159-169 (1962) Marotta, S.F., D.M. Lanuza, L.G. Hiles: Diurnal variations in plasma corticosterone and cations of male rats on two lighting schedules. Horm.Metab.Res. 6: 329-331 (1974) Marotta, S.F., M. Linwong: Excretion of urinary 17-ketosteroids and 17-ketogenic steroids. I. Effeets of age, time of day and season. Chiengmai Med. BuH. 5: 167-181 (1966) Perkoft. G. T., K. Eik-Nes, C.A. Nugent, H.L. Fred, R.A. Nimer, L. Rush, L. T. Samuels, F.H. Tyler: Studies of the diurnal variations of plasma 17-hydroxycorticosteroids in man. J.c1in.Endocr. 19: 432-443 (1959) Rapoport, M.I., R.D. Feigin, J. Bruton, W.R. Beisel: Circadian rhythm for tryptophan pyrrolase activity and its circulating substrate. Science 153: 1642-1644 (1966) Retiene, K., F. Schulz: Circadian rhythmicity of hypothalamic CRH and its eentral nervous regulation. Horm. Metab.Res. 2: 221-224 (1970) Samuels, L. T., H. Brown, K. Eik·Nes, F.H. Tyler, O. V. Dominguez: Extradrenal factors affeeting the levels of 17hydroxycorticosteroids in plasma. Hormones in Blood, ed. by G.E. Wolstenholme and E.C.P. Miliar. Little, Brown & Co., Boston (1957) 208-230 Scapagnini, U., G.P. Moberg, G.R. VanLoon, J. deGroot, W.F. Ganong: Relation of brain 5-hydroxytryptamine content to the diurnal variation in plasma corticosterone in the rat. Neuroendocrinology 7: 90-96 (1971) Silah, J. G.: Ring A reduction of cortisol by hypertensive rat liver in vitro. Endocrinology 88: 1071-1074 (1971) Stevens, W., D.J. Reed, S. Erickson, B.I. Grosser: The binding of corticosterone to brain proteins: Diurnal variation. Endocrinology 93: 1152-1156 (1973) Ungar, F., F. Halberg: Circadian rhythm in the in vitro response of mouse adrenal to adrenocorticotropic hormone. Seienee 137: 1058-1060 (\962) Urquhart, J., F.t:. Yates, A.L. Herbst: Hepatic regulation of adrenal cortical funetion. Endocrinology 64: 816-830 (\959) Van Cantfort, J.: Circadian rhythm of cholesterol-70i-hydroxylase aetivity in geneticaHy blind rats. J.interdiscipI.Cyc1e Res. 5: 89-94 (1974)
RelJuests for reprints should be addressed to: S.F. Marotta, Research Resourees Center, Graduate College (Rm. E-6 MSA), University of lIIinois at the Medical Center, P.D. Box 6998, Chieago, lIIinois 60680, U.S.A.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Referßnees
A ndre ws, R. V.: Temporal seeretory responses of cultured hamster adrenals. Comp.Bioehem.Physiol. 26: 179-193 (1968) Boonayathap, U., S.F. Marotta: Renal handling of cortisol in dogs. l. Free flow studies. Horm.Metab.Res. 6: 74-78 (1974) Cohn, c., D. Joseph, F. Larin, W.J. Shoemaker, R.J. Wurtman: Influenee of feeding habits and adrenal cortex on diurnal rhythm of hepatie tyrosine transaminase activity. Proe.Soe.exp.Bio!. (N.Y.) 133: 460-462 (1970) DeLacerda, L., A. Kowarski, c.J. Migeon: Diurnal variation of the metabolie c1earanee rate of cortiso!. Effeet on measurement of cortisol production rate. J.elin.Endocr. 36: 1043-1049 (1973) Friedman, A.H., C.A. Walker: Circadian rhythms in rat midbrain and caudate nuc1eus biogenie amine levels. J.Physial. (Lond.) 197: 77-85 (1968) Gallagher, T.F., K. Yoshida, H.D. Roffwarg, D.K. Fukushima, E.D. Weitzman, L. Hellman: ACTH and cortisol secretory patterns in man. J.c1in.Endocr. 36: 1058-1068 (1973) Glenn, E.M., R.O. Stafford, S.c. Lyster, B.J. Bowman: Relation between biological activity of hydrocortisone analogues and their rates of inactivation by rat liver enzyme systems. Endocrinology 61: 128-142 (1957) Guillemin, R., G. W. Clayton, H.S. Lipscomb, J.D. Smith: Fluorometric measurement of rat plasma and adrenal corticosterone concentration. J.Lab.clin.Med. 53: 830832 (1959) Hagen, A.A., R.C. Troop: Influence of age, sex, and adrenocortical status on hepatic reduction of cortisone bl vitro. Endocrinology 67: 194-203 (1960) Herbst, A.L., F.E. Yates, D. W. Glenister, J. Urquhart: Variations in hepatic inactivation of corticosterone with changes in food intake: An explanation of impaired corticosteroid metabolism following noxious stimuli. Endocrinology 67: 222-238 (1960) Hiroshige, T., M. Sakakura, S. Itoh: Diurnal variation of corticotropin-releasing activity in the rat hypothalamus. Endocr.japon. 16: 465-469 (1969) Komei, L.: Corticosteroids in human blood. V. Extra-adrenal effect of ACTH upon metabolism of cortiso!. Steroidologia 1: 225-245 (1970) Lanuza, D.M., S.F. Marotta: Circadian and basal interrelationships of plasma cortisol and cations in women. Aerospace Med. 45: 864-868 (974)
Hanruz, P.E., O'Dea, R.F. and N.D. Goldberg: Phosphodiesterase inhibition by papaverine and structurally related compound5. Biochem.Pharmacol. 21: 2266-2268 (1972) Ho, R.J. and E. W. Sutherland: Formation and release of a hormone antagonist by rat adipocytes. J.BioI.Chem. 246: 6822-6827 (1971) Ingebretsen, c., J.F. Clark, D.O. Allen and J. Ashmore: Effect of glucagon, dibutyryl cyclic 3',5'-AMP and phosphodiesterase inhibitors on rat liver phosphorylase activity and cyclic 3',5'-AMP levels. Biochem.Pharmacol. 23: 2139-2146 (1974) Ingebretsen, W.R., Jr. and S.R. Wagle: A rapid method for the isolation of large quantities of rat liver parenchymal cells with high anabolie rates. Biochem.Biophys.Res. Comm. 47: 403-410 (1972) Johnson, M.E.M., N.M. Das, F.R. Butcher and J.N. Fain: The regulation of gluconeogenesis in isolated rat liver eells by glucagon, insulin, dibutyryl cyclic adenosine monophosphate and fatty acids. J.BioI.Chem. 247: 3229· 3235 (1972)
Kono, T.: Destruction and restoration of the insulin effector system of isolated fat cells. J.BioI.Chem. 244: 57775784 (1969) Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. RandalI: Protein measurement with the folin phenol reagent. J.BioI.Chem. 193: 265-275 (1951) Manganiello, V.C., F. Murad and M. Vaughan: Affects of lipolytic and antilipolytic agents on cyclic 3',5'-adenosine monophosphate in fat cells. J.BioI.Chem. 246: 21952202 (1971) Pock, G. and W.R. Kukovetz: Papaverine-induced inhibition of phosphodiesterase activity in various marnmalian tissue. Life Sei. I. Physiol.Pharmacol. 10: 133-144 (1971) fohl, S.L., H.M.J. Krans, L. Birnbaumer and M. Rodbell: Inactivation of glucagon by plasma membranes of rat liver. J.BioI.Chem. 247: 2295-2301 (1972) Sutherland, E. W. and T. W. Rall: Fractionation and characterization of a cyclic adenosine ribonucleotide formed by tissue particles. J.BioI.Chem. 232: 1077-1091 (1958)
Requests for reprints should be addressed to: Dr. D.O. Allen, Department of Pharmacology, Indiana University, School of Medicine, 1100 West Michigan Street, Indianapolis, Indiana 46202 (USA)
Horm. Metab. Res. 7 (1975) 334-337
© Georg Thieme Verlag Stuttgart
The Relation of Hepatic In Vitro I nactivation of Corticosteroids to the Circadian Rhythm of Plasma Corticosterone* S.F. Marotta, L.G. Hiles, D.M. Lanuza and U. Boonayathap** Research Resources Center and the Department of General Nursing, University of Illinois at the Medical Center, Chicago, Illinois, U.S.A.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
334
Summary
Introduction
Plasma corticosterone levels and the in vitro capacity of the liver to reduce the 6,4 3-ketone group of corticosterone were ascertained at 4 hr intervals in male rats maintained on a normal lighting schedule (12L: 120). The rates of 6,4 3-ketone reduction, as weil as wet liver weight, were highest during the early portion (08.00 hr) of the light period when liver protein and plasma corticosterone concentrations were low. Shortly (2000 hr) after the beginning of the dark period plasma corticosterone reached peak levels, while hepatic inactivation of corticosterone was markedly depressed. This inverse relationship suggests that the rhythmicity in the capacity of the liver to inactivate corticosterone may contribute to the circadian periodicity of plasma corticosterone.
Although circadian rhythms in plasma adrenocortical steroid concentrations have been weH documented in human beings (Samuels, Browll, Eik-Nes, Tyler and Dominguez 1957, Lanuza and Marotta 1974) and lower animals (Ungar and Halberg 1962, Hiroshige, Sakakura and ftoh 1969, Marotta, Lanuza and Hiles 1974), investigators continue to search at various levels of the hypothalamo-hypophyseal-adrenocortical (HHA) system for the mechanisms which initiate or affect this periodicity. Thus, circadian r:-,ythms have been reported for a~ the uptake of corticosterone (lIß, 21-dihydroxy-4-pregnene-3,20-dione) by the hippocampus which is considered a possible negative feedback site (Stevens, Reed, Erickson and Grosser 1973), b) proposed neurotransmitters (biogenic amines) for the corticotropin-releasing hormone (CRH) in the central nervous system (Friedman and Walker 1968, Scapagnini, Moberg, VanLoon, DeGroot and Ganong 1971), c) hypothalamic content of CRH (Hiroshige, Sakakura and ftoh 1969, Retiene and Schulz 1970), d) concentrations of adrenocorticotropin (ACTH) in the adenohypophysis and plasma
Key·Words: Circadian Plasma Corticosterone - Hepatic Steroid Inactivation
·Supported in part by internal grants of the University of lllinois and by the Office of Naval Research contract NR 201-020 "Present address: Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand Received: 13 Jan. 1975
Accepted: 21 Apr. 1975
335
(Retiene and Schulz 1970, Gallagher, Yoshida, Rolf warg, Fukushima, Weitzman and Hel/man 1973), e) in vivo and in vitro responses of the adrenal cortex to the administration of ACTH (Ungar and Halberg 1962, Andrews 1968) and f) in vitro production of steroids by adrenal quarters in the absence of ACTH (Ungar and Halberg 1962, Andrew 1968).
at 06.00 hr from the cages of animals sacrificed from 08.00 to 16.00 hr, and at 18.00 hr for those sacrificed from 20.00 to 04.00 hr. The animals were decapitated in less than 15 seconds after removal from their cages in the constant temperature room. Trunk blood, which was collected in heparinized tubes, was immediately centrifuged and the plasma stored at -20 oC until analyzed for corticosterone by the fluorometric method of Guillemin, Clayton, Lipscomb and Smith (1959).
The periodicity in plasma steroid levels is dependent upon not only those HHA mechanisms which govern the cyclic release of adrenocortical steroids, bu t also those mechanisms wh ich affect the metabolism and renal excretion of steroids. Circadian variations in the latter have been correlated with the diurnal changes in glomerular filtration rate (Markei, Sharp, Slorach and Vipond 1962, Marotta and Linwong 1966), which in turn may influence the amounts of steroid found in urine (Boonayathap and Marotta 1974). Studies on whole body metabolism of adrenocortical steroids, as assessed by the plasma disappearance rate (t 1/2) following rapid infusion of pharmacologic loads of cortisol (IIß, 17, 21-trihydroxy-4-pregnene-3, 20-dione) have not shown diurnal differences (Perkol!. Eik-Nes, Nugent, Fred. Nimer, Rush, Samuels and Tyler 1959). On the other hand, deLacerda, Kowarski and Migeon (1973), using radio labelled cortisol, have reported diurnal variations in the metabolic clearance rate of this steroid. Since Samuels et al. (1957) suggested that the t 1/2 is affected by the initial cortisol concentration, one could postulate that the circadian alterations in plasma cortisol levels would also modify the t 1/2 of this steroid. However, the disappearance of cortisol from plasma is affected primarily by the rate at which this steroid is inactivated by the liver such that the in vitro enzymatic activity (e.g. t,4 -steroid hydrogenase ) of the Iiver is closely associated with the in vivo changes in t 1/2 (Herbst, Yates, Glenister and Urquhart 1960). Although the rates of hepatic inactivation of adrenocortical steroids have been shown to vary when animals are subjected to various stressors (Herbst et al. 1960, Silah 1971), no attempt has been made to ascertain whether the liver exhibits a circadian periodicity in its enzymatic capacity to inactivate these steroids. Thus a study was designed to a) determine the in vitro rate of reduction of corticosterone by the liver of male rats sacrificed every 4 hr throughout a 24 hr period, and b) correlate this reduction rate with the weil known circadian rhythm in plasma corticosterone levels.
The entire liver was rapidly removed from the animals and placed in ice cold iso tonic saline. The Iiver was biotted dry, weighed and a portion (approximately 500 mg) homogenized in a Sorval Omni-Mixer with a solution containing sucrose (0.25 M) and nicotinamide (0.04 M) such that the final concentration of the homogenate was 20% w/v. The homogenate was centrifuged at 2000 RPM for 15 min at 4 0 C. Protein concentration (Lowry, Rosebrough, Fa" and Randall 1951) and the capacity of the liver to inactivate corticosterone. as measured by the reduction rate of the t,4 3ketone group (Glenn, Stafford, Lyster and Bowman 1957), were determined in the supernatant fraction. All the necessary cofactors (magnesium chloride, phosphate buffer at pH 7.4. sucrose, nicotinamide, glucose-6-phosphate, trisodium iso citrate and nicotinamide adenine dinucleotide phosphate) as prescribed by Glenn et al. (1957) were present in the incubation medium together with 100 pg corticosterone which served as the substrate. After one hr incubat ion at 370 C the homogenate was extracted with purified cold methylene chloride and the Iatter phase was washed successively with 0.1 N NaOH, 0.1 N HCI and water. Aliquots of the methylene chloride extract were evaporated to dryness at 35 0 C under reduced pressure (-10 Ib/sq.in). The residue was dissolved in 5.0 ml methyl alcohol, transferred to matched quartz cuvettes and read at 240 nm in a Beckman DBG spectrophotometer. Appropriate tissue blanks incubated at OOC and standards were also carried throughout the entire procedure.
Materials and Methods Forty-two male rats (Holtzman Co., Madison, Wis.) were placed 7/cage in a constant temperature room (21 oC) for approximately two weeks with a lighting schedule of 12L: 12D with light from 06.00 to 18.00 hr. Seven rats were sacrificed by decapitation every 4 hr beginning at 08.00 hr and ending the following morning at 04.00 hr. Food (Purina rat chow), which had been fed ad libitum, was removed
Results The mean body weights of rats sacrificed at 4 hr intervals throughout the day were not significantly different from each other, while the liver weights (gm/IOO gm B.W.) of rats sacrificed frorn 08.00 to 16.00 hr were significantly heavier (10 to 45%) than those found in animals from 20.00 to 04.00 hr (Table 1). These changes in liver weight were probably due to changes in water content, since soon after the beginning (06.00 hr) of the light period the hepatic protein concentrations were considerably lower (110 to 115 mg/gm L.W.) than the peak values (137 to 144 mg/gm L.W.) noted toward the end of the dark period. In general, the capacity of the liver to reduce the t,4 3-ketone group, whether reported as the rate of corti~osterone disappearance per gram wet liver (f..Lg Cort./gm L.W./hr) or per gram liver protein (mg Cort./gm L.P./hr), was significantly greater during the light (06.00 to 18.00 hr) than during the dark (18.00 to 06.00 hr) period. The capacity of the liver to inactivate corticosterone began to gradually decline from peak disappearance rates (1.63 mg/gm L.P./hr) at 08.00 hr to minimal rates (0.77 mg/gm L.P./hr) at 24.00 hr before beginning to rise in the
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Hepatic In Vitro Inactivation of Corticosteroids
336
S.F. Marotta, L.G. Hiles, D.M. Lanuza and U. Boonayathap
Table 1. Relation of plasma corticosterone levels to hepatic weight, pro tein content and 6 4 3-ketone reduction rate of male rats on a schedule of light from 06.00 to 18.00 hr and dark from 18.00-06.00 hr Time of Day (hr) 12.00
16.00
20.00
24.00
04.00
Body Weight (B.W.) gm
227'" (213-242)
217 (204-231)
233 (224-243)
227 (221-234)
215 (201-228)
228 (219-238)
Liver Weight (L.W.) gm/IOO gm B.W.
4.9 (4.6-5.3)
4.8 (4.6-5.0)
4.5 (4.3-4.6)
3.9 (3.6-4.1 )
3.8 (3.7-3.9)
3.3 (3.2-3.4 )
6 4 3-Ketone Reduction
174 (165-182)
175 (150-200)
165 (154-178)
118 (106-130)
106 (90-123)
118 (75-163)
UO (100-120)
115 (111-119)
131 (128-135)
133 (130-135)
137 (132-142)
144 (140-149)
mg Cort./gm L.P./hr
1.63 (1.48-1.78)
1.55 (1.31-1.79)
1.25 (1.17-1.33)
0.90 (0.81-0.98)
0.83 0.77 (0.62-0.91 ) (0.53-1.13 )
Plasma Corticosterone pg/lOO ml
5.0 (3.3-6.8)
5.6 (4.3-6.9)
14.2 (11.9-16.6)
21.7 (15.2-28.1)
14.3 (10.5-18.0)
Cort./gm L.W./hr Liver Protein (L.P.) mg/gm L.W.
l1g
6 4 3-Ketone Reduction
7.9 (6.2-9.7)
"'Mean with 95% confidence limits in ( ).
next 4 hr (Table l). During this time the plasma corticosterone pattern, which exhibited basal levels (5.0 to 5.9 /Jg/loo ml) in the morning and significantly higher levels (14.2 to 2l.7 /Jg/lOO ml) from 16.00 to 24.00 hr, were inversely (r = 0.51; P < 0.05) related to the rates of hepatic inactivation of corticosterone. Although the time (08.00 to 12.00 hr) for minimal plasma corticosterone levels and maximal hepatic inactivation coincided, peak corticosterone levels (20.00 hr) were observed approximately 4 hr prior to the nadir of hepatic in vitro inactivation of corticosterone.
inactivation of steroids may not only be mediated via the release of adrenocortical steroids, but also to its direct effect on inhibiting the hepatic enzymes involved in reducing ring A of the corticosteroids
(Komel 1970).
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Measurements
In addition to the above factors which affect the hepatic inactivation of adrenocortical steroids, Herbst et al. (1960) showed that variations in food intake markedly affect the activity of this hepatic enzyme system, and hence any stressor which diminishes food intake depresses the capacity of the liver to inactivate adrenocortical steroids. Diurnal studies on food intake have shown that rats on a normal lightDiscussion ing schedule consume more food from 18.00 to Many investigators have reported circadian variations 24.00 hr than at other times during the day (Herbst in the activities of hepatic enzymes involved in the et al. 1960). To partially avoid this factor in the metabolism of cholesterol (VanCantfort 1974), tyro- present study, rats sacrificed from 08.00 to 16.00 hr si ne (Cohn, Joseph, Larin, Shoemaker and Wurtman did not have access to food from 06.00 hr, while those 1970) and tryptophan (Rapoport, Feigin, Bruton sacrificed from 20.00 to 04.00 hr were withou t food and Beisel 1966). Furthermore, these enzymatic acfrom 18.00 hr. When comparing groups of rats durtivities are related to the circulating levels of adreno- ing the light period with those du ring the dark period cortical steroids since adrenalectomy modified their but without access to food for the same length of circadian periodicity. Hypo- and hyperadrenocortitime, the rates of reduction of the 6 4 3-ketone group were always higher during the light period than thc calism also appear to affect the hepatic capacity to dark period. It is unlikely, therefore, that the periodreduce the 6 4 _3 ketone group. Urquhart, Yates and Herbst (1959) have shown that severe adrenocortiicity observed in hepatic inactivation of corticosterone cal insufficiency in rats with an accompanying loss is markedly affected by the circadian rhythm in food in body weight caused a marked depression in hepatic intake, since minimal hepatic activity was observed reduction rates, while unilateral adrenalectomy with- during aperiod of normally high food intake. out a loss in body weight or bilateral adrenalectomy The data presented herein suggest that the sustained in rats maintained on saline (Hagen and Troop 1960), elevation in plasma corticosterone levels, and presum~aused no c~ange or a ~light in.cre~e, respect.ively, ably ACTH as weIl, which preceded minimal in vitro m the capacIty of the hver to machv.ate corhcost~rone. hepatic inactivation of corticosterone, may contribute On ~he other hand, pretreatment ~f l~tact rats ~lt.h to the depression in hepatic enzymatic activity; and cOr~lSO?e or ACT~ depre~sed the zn vltro hepahc m- in turn the latter would then cause a gradual increase in plasma corticosterone levels which would inhibit actlvahon of corhcosterOlds (Urquhart, Yates an~ Herbst 1959, Hagen and Troop 1960). Recent eVlthe release of ACTH. Thus, it is conceivable that the dence suggests that the effects of ACTH on hepatic
Hepatic In Vitro Inactivation of Corticosteroids
daily variations observed in plasma eortieosteroid eoneentrations may not only be due to rhythms previously shown in hypothalamie CRH (JIiroshige, Sakakura and Itoh 1969), adenohypophyseal and plasma ACTH (Retiene and Schulz 1970, Gal/agher
337
et al. 1973), adrenoeortieal seeretion (Andrews 1968), and the metabolie clearanee of eortieosteroids (de Lacerda, Kowarski and Migeon 1973), but also to the diurnal eapaeity of the liver to inaetivate these steroids. Lowry, O.H., N.J. Rosebrough, A.L. Farr, R.F. Randall: Protein measurement with Folin-phenol re agent. J.biol. Chem. 193: 265-275 (1951) Martel, P.J., G. W.G. Sharp, S.A. Slorach, H.J. Vipond: A study of the roles of adrenocortical steroids and glomerular filtration rate in the mechanism of the diurnal rhythm of water and electrolyte excretion. J.Endocr. 24: 159-169 (1962) Marotta, S.F., D.M. Lanuza, L.G. Hiles: Diurnal variations in plasma corticosterone and cations of male rats on two lighting schedules. Horm.Metab.Res. 6: 329-331 (1974) Marotta, S.F., M. Linwong: Excretion of urinary 17-ketosteroids and 17-ketogenic steroids. I. Effeets of age, time of day and season. Chiengmai Med. BuH. 5: 167-181 (1966) Perkoft. G. T., K. Eik-Nes, C.A. Nugent, H.L. Fred, R.A. Nimer, L. Rush, L. T. Samuels, F.H. Tyler: Studies of the diurnal variations of plasma 17-hydroxycorticosteroids in man. J.c1in.Endocr. 19: 432-443 (1959) Rapoport, M.I., R.D. Feigin, J. Bruton, W.R. Beisel: Circadian rhythm for tryptophan pyrrolase activity and its circulating substrate. Science 153: 1642-1644 (1966) Retiene, K., F. Schulz: Circadian rhythmicity of hypothalamic CRH and its eentral nervous regulation. Horm. Metab.Res. 2: 221-224 (1970) Samuels, L. T., H. Brown, K. Eik·Nes, F.H. Tyler, O. V. Dominguez: Extradrenal factors affeeting the levels of 17hydroxycorticosteroids in plasma. Hormones in Blood, ed. by G.E. Wolstenholme and E.C.P. Miliar. Little, Brown & Co., Boston (1957) 208-230 Scapagnini, U., G.P. Moberg, G.R. VanLoon, J. deGroot, W.F. Ganong: Relation of brain 5-hydroxytryptamine content to the diurnal variation in plasma corticosterone in the rat. Neuroendocrinology 7: 90-96 (1971) Silah, J. G.: Ring A reduction of cortisol by hypertensive rat liver in vitro. Endocrinology 88: 1071-1074 (1971) Stevens, W., D.J. Reed, S. Erickson, B.I. Grosser: The binding of corticosterone to brain proteins: Diurnal variation. Endocrinology 93: 1152-1156 (1973) Ungar, F., F. Halberg: Circadian rhythm in the in vitro response of mouse adrenal to adrenocorticotropic hormone. Seienee 137: 1058-1060 (\962) Urquhart, J., F.t:. Yates, A.L. Herbst: Hepatic regulation of adrenal cortical funetion. Endocrinology 64: 816-830 (\959) Van Cantfort, J.: Circadian rhythm of cholesterol-70i-hydroxylase aetivity in geneticaHy blind rats. J.interdiscipI.Cyc1e Res. 5: 89-94 (1974)
RelJuests for reprints should be addressed to: S.F. Marotta, Research Resourees Center, Graduate College (Rm. E-6 MSA), University of lIIinois at the Medical Center, P.D. Box 6998, Chieago, lIIinois 60680, U.S.A.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Referßnees
A ndre ws, R. V.: Temporal seeretory responses of cultured hamster adrenals. Comp.Bioehem.Physiol. 26: 179-193 (1968) Boonayathap, U., S.F. Marotta: Renal handling of cortisol in dogs. l. Free flow studies. Horm.Metab.Res. 6: 74-78 (1974) Cohn, c., D. Joseph, F. Larin, W.J. Shoemaker, R.J. Wurtman: Influenee of feeding habits and adrenal cortex on diurnal rhythm of hepatie tyrosine transaminase activity. Proe.Soe.exp.Bio!. (N.Y.) 133: 460-462 (1970) DeLacerda, L., A. Kowarski, c.J. Migeon: Diurnal variation of the metabolie c1earanee rate of cortiso!. Effeet on measurement of cortisol production rate. J.elin.Endocr. 36: 1043-1049 (1973) Friedman, A.H., C.A. Walker: Circadian rhythms in rat midbrain and caudate nuc1eus biogenie amine levels. J.Physial. (Lond.) 197: 77-85 (1968) Gallagher, T.F., K. Yoshida, H.D. Roffwarg, D.K. Fukushima, E.D. Weitzman, L. Hellman: ACTH and cortisol secretory patterns in man. J.c1in.Endocr. 36: 1058-1068 (1973) Glenn, E.M., R.O. Stafford, S.c. Lyster, B.J. Bowman: Relation between biological activity of hydrocortisone analogues and their rates of inactivation by rat liver enzyme systems. Endocrinology 61: 128-142 (1957) Guillemin, R., G. W. Clayton, H.S. Lipscomb, J.D. Smith: Fluorometric measurement of rat plasma and adrenal corticosterone concentration. J.Lab.clin.Med. 53: 830832 (1959) Hagen, A.A., R.C. Troop: Influence of age, sex, and adrenocortical status on hepatic reduction of cortisone bl vitro. Endocrinology 67: 194-203 (1960) Herbst, A.L., F.E. Yates, D. W. Glenister, J. Urquhart: Variations in hepatic inactivation of corticosterone with changes in food intake: An explanation of impaired corticosteroid metabolism following noxious stimuli. Endocrinology 67: 222-238 (1960) Hiroshige, T., M. Sakakura, S. Itoh: Diurnal variation of corticotropin-releasing activity in the rat hypothalamus. Endocr.japon. 16: 465-469 (1969) Komei, L.: Corticosteroids in human blood. V. Extra-adrenal effect of ACTH upon metabolism of cortiso!. Steroidologia 1: 225-245 (1970) Lanuza, D.M., S.F. Marotta: Circadian and basal interrelationships of plasma cortisol and cations in women. Aerospace Med. 45: 864-868 (974)
